Current treatments for depression have limitations that significantly impact treatment outcomes. One is that a large percentage (~40%) of patients are resistant to any current treatment. Evidence now indicates that the endogenous opioid system may represent a previously under-appreciated system that can yield novel anti-depressant treatments. We will extend recent preliminary data to test this possibility directly. We will first test the hypothesis that deletion of proenkephalin-derived peptides alters the behavioral and molecular responses to chronic fluoxetine. We have recently demonstrated that the known antidepressant tianeptine is an agonist of the mu opioid receptor (MOR). We and others have also found that expression of the proenkephalin (Penk) gene, which encodes a precursor to an endogenous MOR ligand, is dramatically up regulated in the dentate gyrus following chronic treatment with several SSRIs. These findings suggest that Penk peptides may be central components in the molecular response to anti-depressant treatment. We will test this possibility by determining whether chronic behavioral anti-depressant activities of fluoxetine are altered or abolished in Penk KO mice. We will further determine the extent to which previously-determined gene expression changes in response to chronic fluoxetine are altered in Penk KO mice and thus begin to position Penk up-regulation in this molecular cascade. Our second aim will test the hypothesis that pharmacologic up-regulation of enkephalin peptides in specific brain regions is sufficient to promote behavioral responses to SSRIs. Systemic injection of enkephalinase inhibitors can produce anti-depressive and analgesic actions resulting from enkephalin peptide up-regulation, though the cellular sites of activity have remained undefined. Our preliminary experiments indicate that RB-101 rapidly increases analgesic activity following icv injection. We will extend these studies to examine acute anti-depressant effects of RB-101 injection directly into several brain regions in both WT and Penk deficient mice. This pharmacologic approach will thus mimic one specific molecular response to chronic SSRI treatment and thus begin to determine whether increased Penk alone is sufficient to mediate anti-depressant responses as well as identify the specific brain regions that can mediate such effects. Finally, we will identify the opioid receptor specificity of increased enkephalin action. Enkephalin can bind the delta opioid receptor (DOR) as well as MOR and these opioid system components are co- expressed in the hippocampus, a major locus implicated in anti-depressant action. We will compare anti- depressive actions of RB-101 in MOR, DOR and MOR/DOR KO mice following injection into multiple brain regions to determine whether responses are altered in the absence of either or both receptors. These studies will thus identify the receptor(s) mediating enkephalin activities and begin to develop a more detailed understanding of the circuitry underlying opioid system activity in anti-depressive action.